Nucleic Acid Molecules Encoding Cyclotide Polypeptides and Methods of Use

ABSTRACT

The present invention relates to isolated nucleic acids encoding plant cyclotides. The invention also relates to the construction of a chimeric gene encoding all or a portion of the plant cyclotides, in sense or antisense orientation, wherein expression of the chimeric gene results in the production of altered levels of plant cyclotides in a transformed host cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalApplication No. 60/575,571, filed May 28, 2004, and U.S. Utilityapplication Ser. No. 11/129,817, filed May 16, 2005, which are herebyincorporated in their entirety by reference.

FIELD OF THE INVENTION

The present invention relates to naturally-occurring and recombinantnucleic acids encoding cyclotides characterized by activity againstplant pathogens. Compositions and methods of the invention utilize thedisclosed nucleic acids, and their encoded polypeptides to control plantpathogens.

BACKGROUND OF THE INVENTION

Plant pathogens are responsible for significant annual crop yieldlosses. One strategy for the control of plant pathogens is the use ofresistant cultivars selected for, or developed by, plant breeders forthis purpose. However, novel mechanisms for pathogen resistance can beimplemented more quickly by molecular methods of crop protection than bytraditional breeding methods. Accordingly, molecular methods are neededto supplement traditional breeding methods to protect plants frompathogen attack.

Plants rely heavily on a chemical and biological armory for theirdefense from a variety of pests and pathogens. Small cystine-richproteins that have been implicated in host defense and isolated fromplant sources include defensins, thionins, and small antimicrobialproteins (AMP's). Cyclotides, also cystine-rich molecules, have recentlybeen recognized and characterized as being involved in host defense(Craik et al. (1999), J. Mol. Biol. 294: 1327-1336; Craik et al. (2000),Toxicon 39: 43-60). Cyclotide polypeptides are encoded by genesequences, are produced as linear precursors, are cystine-rich, and arecapable of being cyclized via a peptide bond. Cyclotides display adiverse range of biological activities such as antibacterial activity,antifungal activity, anti-HIV activity, and uterotonic activity (Craik(2001), Toxicon 39: 1809-1813). Cyclotides have additionally been shownto possess insecticidal activity (Jennings et al. (2001) Proc. Natl.Acad. Sci. U.S.A. 98:10614-10619). Cyclized cyclotides differ fromclassical proteins in that they have no free N- or C-terminus due totheir amide-circularized backbone.

Cyclotide polypeptides are derived from longer precursor proteins andthus both cleavage and cyclization steps are involved in the productionof the cyclic backbone. The cyclic backbone of the cyclotide moleculetypically ranges in size from 29 to 37 amino acid residues and has threedisulfide bonds that form a cystine knot motif where two disulfide bondsand their connecting backbone strands form a ring that is threaded bythe third disulfide bond. The mechanism(s) inherent to backbonecyclization is currently not known. One possibility is enzymatic orchemical involvement in both the backbone cleavage of the mature domainand the subsequent cyclization. The combined features of the cycliccystine knot produces a unique protein fold that is topologicallycomplex and has exceptional chemical and biological stability.

The majority of the plant cyclotides have been isolated from Rubiaceaeand Violaceae plants (Gustafson et al. (1994), J. Nat. Prod. 116:9337-9338; Gustafson et al. (2000), J. Nat. Prod. 63: 176-178; Witherupet al. (1994), J. Nat. Prod. 57: 1619-1625; Saether et al. (1995),Biochemistry 34, 4147-4158; Bokesch et al. (2001), J. Nat. Prod. 64:249-250; Schöpke et al. (1993), Sci. Pharm. 61: 145-153; Claeson et al.(1998), J. Nat. Prod. 61: 77-81; Göransson et al. (1999), J. Nat. Prod.62: 283-286; Hallock et al. (2000), J. Org. Chem. 65: 124-128;Broussalis et al. (2001), Phytochemistry 58: 47-51). Recently, twomembers of a new sub-class of the cyclotide family have been discoveredin Curcurbitaceae (Hernandez et al. (2000), Biochemistry 39: 5722-5730;Felizmenio-Quimio et al. (2001), J. Biol. Chem. 276: 22875-22882; Heitzet al. (2001), Biochemistry 40: 7973-7983; Trabi and Craik, (2002),Trends in Biochem. Sci. 27: 132-138).

Cyclotides may be used in transgenic plants in order to produce plantswith increased resistance to pathogens such as fungi, viruses, bacteria,nematodes, and insects. Thus, embodiments of the present inventionsolves needs for the enhancement of a plant's defensive response via amolecularly based mechanism which can be quickly incorporated intocommercial crops.

SUMMARY OF THE INVENTION

Compositions and methods relating to pathogen resistance are provided.

The cyclotide sequences of the embodiments find use in enhancing theplant pathogen defense system. The compositions and methods of theembodiments can be used for enhancing resistance to plant pathogensincluding fungal pathogens, plant viruses, microorganisms, nematodes,and the like. The method involves stably transforming a plant with anucleotide sequence capable of modulating the plant pathogen defensesystem operably linked with a promoter capable of driving expression ofa gene in a plant cell.

Transformed plants, plant cells, and seeds, as well as methods formaking such plants, plant cells, and seeds, are additionally provided.It is recognized that a variety of promoters will be useful in theembodiments of the invention, the choice of which will depend in partupon the desired level of expression of the disclosed genes. It isrecognized that the levels of expression can be controlled to modulatethe levels of expression in the plant cell.

Embodiments of the present invention are directed to cyclizablemolecules and their linear precursors; cyclic peptides, polypeptides orproteins; and additionally include linear forms of non-cyclic structuralhomologues of the cyclic peptides, polypeptides and proteins. Alsoincluded are derivative forms of the cyclized molecules and their linearprecursors encoded by the subject nucleic acid molecules. The cyclic andlinear peptides, polypeptides or proteins may be naturally occurring ormay be modified by the insertion or substitution of heterologous aminoacid sequences.

One embodiment of the present invention provides an isolated nucleicacid molecule comprising a sequence of nucleotides, which sequence ofnucleotides, or its complementary form, encodes an amino acid sequenceor a derivative form thereof capable of being cyclized within a cell ora membrane of a cell to form a cyclic backbone wherein the cyclicbackbone comprises sufficient disulfide bonds to confer a stabilizedfolded structure on the three dimensional structure of the backbone. Theamino acid sequence may also be cyclizable in an in vitro systemcomprising, for example, cyclizing enzymes or a chemical means forcyclization.

The embodiments also extend to the peptide, polypeptide or proteinsequences which are capable of cyclizing in the absence of any otherexogenous factor and more specifically capable of circularizing througha catalytic process being an inherent activity of the peptides,polypeptides or proteins.

The embodiments comprise a peptide sequence that can be processed from alarger polypeptide sequence. More specifically, the embodiments refer toa peptide sequence, which can be cleaved and cyclized. The embodimentsfurther extend to linear forms and precursor forms of the peptide,polypeptide or protein, which may also have activity or other utilities.The embodiments additionally extend to engineering crop plants with thesequences of the embodiments in order to produce plants that areresistant to pathogens.

A further embodiment of the present invention provides an isolatednucleic acid molecule comprising a sequence of nucleotides, whichsequence of nucleotides, or its complementary form, encodes an aminoacid sequence or a derivative form thereof capable of forming astructural homologue of a cyclic peptide, polypeptide, or protein withina cell or a membrane of a cell to form a backbone wherein the backbonecomprises sufficient disulfide bonds to confer a stabilized foldedstructure on the three-dimensional structure of the backbone wherein thebackbone comprises free amino and carboxy termini.

The embodiments include an isolated polynucleotide comprising anucleotide sequence selected from the group consisting of: a nucleotidesequence set forth in SEQ ID NO: 1 and 3; a nucleotide sequence thatencodes a polypeptide having the amino acid sequence set forth in SEQ IDNO: 2 and 4; a nucleotide sequence characterized by at least 85%sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1and 3; a nucleotide sequence characterized by at least 90% sequenceidentity to the nucleotide sequence set forth in SEQ ID NO: 1 and 3; anucleotide sequence characterized by at least 95% sequence identity tothe nucleotide sequence set forth in SEQ ID NO: 1 and 3; and anucleotide sequence that comprises the complement of any one of theabove.

The embodiments also relate to a chimeric gene comprising an isolatedpolynucleotide of the embodiments operably linked to suitable regulatorysequences.

A further embodiment concerns an isolated host cell comprising achimeric gene or an isolated polynucleotide of the embodiments. The hostcell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic,such as a bacterial cell. The embodiments also relate to a virus,preferably a baculovirus, comprising a chimeric gene or an isolatedpolynucleotide of the embodiments.

The embodiments further provide a process for producing an isolated hostcell comprising a chimeric gene or an isolated polynucleotide of theembodiments, the process comprising either transforming or transfectingan isolated compatible host cell with a chimeric gene or isolatedpolynucleotide of the embodiments.

The embodiments provide an isolated polypeptide selected from the groupconsisting of: a polypeptide comprising an amino acid sequence set forthin SEQ ID NO: 2 and 4; a polypeptide characterized by at least 97%identity to SEQ ID NO: 2 and 4; a polypeptide characterized by at least98% identity to SEQ ID NO: 2 and 4; and a polypeptide characterized byat least 99% identity to SEQ ID NO: 2 and 4.

The embodiments additionally provide a method for impacting a plantpathogen comprising introducing into a plant or cell thereof at leastone nucleotide construct comprising a coding sequence operably linked toa promoter that drives expression of a plant cyclotide polypeptide inplant cells, wherein said nucleotide sequence is selected from the groupconsisting of: a nucleotide sequence set forth in SEQ ID NO: 1 and 3; anucleotide sequence that encodes a polypeptide having the amino acidsequence set forth in SEQ ID NO: 2 and 4; a nucleotide sequencecharacterized by at least 85% sequence identity to the nucleotidesequence set forth in SEQ ID NO: 1 and 3; a nucleotide sequencecharacterized by at least 90% sequence identity to the nucleotidesequence set forth in SEQ ID NO: 1 and 3; a nucleotide sequencecharacterized by at least 95% sequence identity to the nucleotidesequence set forth in SEQ ID NO: 1 and 3; and a nucleotide sequence thatcomprises the complement of any one of the above.

Expression cassettes and stably transformed plants are also provided bythe embodiments. The polypeptides of the embodiments are useful inprotecting plants from various pests including, but not limited to,fungi, bacteria, viruses and nematodes.

The embodiments provide nucleic acids and fragments and variants thereofwhich encode polypeptides or mature polypeptides that possess activityagainst plant pathogens. In some embodiments, the nucleotide sequencesencode polypeptides that are pesticidal against nematodes. In otherembodiments, the nucleotide sequences encode polypeptides that areactive against fungal pathogens.

In a particular embodiment, a transformed plant can be produced using anucleic acid that has been optimized for increased expression in a hostplant. For example, the cyclotide polypeptides of the embodiments can beback-translated to produce nucleic acids comprising codons optimized forexpression in a particular host, for example a crop plant such as asoybean plant. In some embodiments are provided transgenic plantsexpressing polypeptides that find use in methods for impacting variousplant pathogens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the HPLC profile of the crude extract from the Viola spp.showing the absorbance measured at 214 nm (b). The region correspondingto the elution times of the plant cyclotides is expanded out (a). Thepeaks were pooled together and purified using a reverse phase capillarycolumn.

FIG. 2 depicts the HPLC profile of the reverse phase capillarypurification of the bioactive cyclotides pooled from the crude extractshown in FIG. 1(a) using the following gradient: 10-30% buffer B for 10minutes followed by 30-60% B over 70 minutes. Each number represents apossible bioactive cyclotide. The nematocidal activity was confined topeak labeled 2, hereafter referred to as cyclotide 2.

DETAILED DESCRIPTION OF THE INVENTION

The embodiments of the present invention provide, inter alia,compositions and methods for modulating the total level of polypeptidesof the embodiments and/or altering their ratios in a plant. As usedherein, the term “modulation” is intended to mean an increase ordecrease in a particular character, quality, substance, or response. Thecompositions comprise nucleotide and amino acid sequences from variousplant species.

The following definitions and methods are provided to better define theembodiments of the present invention and to guide those of ordinaryskill in the art in the practice of the embodiments. Unless otherwisenoted, terms are to be understood according to conventional usage bythose of ordinary skill in the relevant art. Definitions of common termsin molecular biology may also be found in Rieger et al., Glossary ofGenetics: Classical and Molecular, 5^(th) edition, Springer-Verlag; NewYork, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.The nomenclature for DNA bases as set forth at 37 CFR §1.822 is used.

As used herein, the term “comprising” means “including but not limitedto”.

As used herein, “cyclotide-like activity” refers to the inhibition ofpathogen growth or damage caused by a variety of pathogens, including,but not limited to, fungi, nematodes, viruses, and bacteria.

As used herein, “antimicrobial” or “antimicrobial activity” refers toantibacterial, antiviral, antinematodal, and antifungal activity.Accordingly, the polypeptides of the embodiments may enhance resistanceto insects and nematodes. Any one cyclotide may exhibit a spectrum ofantimicrobial activity that may involve one or more antibacterial,antifungal, antiviral, insecticidal, antinematodal, or antipathogenicactivities.

As used herein, the terms “plant pathogen” or “plant pest” refer to anyorganism that can cause harm to a plant. A plant can be harmed by aninhibition or slowing of the growth of a plant, by damage to the tissuesof a plant, by a weakening of the immune system of a plant, by areduction in the resistance of a plant to abiotic stresses, by apremature death of the plant, and the like. Plant pathogens and plantpests include, but are not limited to nematodes, and organisms such asfungi, viruses, and bacteria.

As used herein, the terms “disease resistance” or “pathogen resistance”are intended to mean that the organisms avoid the disease symptoms thatare the outcome of organism-pathogen interactions. That is, pathogensare prevented from causing diseases and the associated disease symptoms,or alternatively, the disease symptoms caused by the pathogen areminimized or lessened.

As used herein, the terms “nematode resistant” and “impacting nematodepests” refer to effecting changes in nematode feeding, growth, and/orbehavior at any stage of development, including but not limited to:killing the nematode; retarding growth; preventing reproductivecapability; and the like.

As used herein, the terms “pesticidal activity” and “anti-nematodalactivity” are used synonymously to refer to activity of an organism or asubstance (such as, for example, a protein) that can be measured by butis not limited to pest mortality, pest weight loss, pest attraction,pest repellency, and other behavioral and physical changes of a pestafter feeding and exposure for an appropriate length of time. Forexample “pesticidal proteins” are proteins that display pesticidalactivity by themselves or in combination with other proteins.

An “antimicrobial agent,” a “pesticidal agent,” an “antiviral agent,” an“anti-nematodal or nematicidal agent,” and/or a “fungicidal agent” willact similarly to suppress, control, and/or kill the invading pathogen.

The term “antipathogenic compositions” is intended to mean that thecompositions of the embodiments have activity against plant pathogens;including fungi, microorganisms, viruses and nematodes, and thus arecapable of suppressing, controlling, and/or killing the invadingpathogenic organism. An antipathogenic composition of the embodimentswill reduce the disease symptoms resulting from pathogen challenge by atleast about 5% to about 50%, at least about 10% to about 60%, at leastabout 30% to about 70%, at least about 40% to about 80%, or at leastabout 50% to about 90% or greater. Hence, the methods of the embodimentscan be utilized to protect organisms, particularly plants, from invadingpathogens.

Assays that measure antipathogenic activity are commonly known in theart, as are methods to quantitate disease resistance in plants followingpathogen infection. See, for example, U.S. Pat. No. 5,614,395, hereinincorporated by reference. Such techniques include, measuring over time,the average lesion diameter, the pathogen biomass, and the overallpercentage of decayed plant tissues. For example, a plant eitherexpressing an antipathogenic polypeptide or having an antipathogeniccomposition applied to its surface shows a decrease in tissue necrosis(i.e., lesion diameter) or a decrease in plant death following pathogenchallenge when compared to a control plant that was not exposed to theantipathogenic composition. Alternatively, antipathogenic activity canbe measured by a decrease in pathogen biomass. For example, a plantexpressing an antipathogenic polypeptide or exposed to an antipathogeniccomposition is challenged with a pathogen of interest. Over time, tissuesamples from the pathogen-inoculated tissues are obtained and RNA isextracted. The percent of a specific pathogen RNA transcript relative tothe level of a plant specific transcript allows the level of pathogenbiomass to be determined. See, for example, Thomma et al. (1998) PlantBiology 95:15107-15111, herein incorporated by reference.

Furthermore, in vitro antipathogenic assays include, for example, theaddition of varying concentrations of the antipathogenic composition topaper disks and placing the disks on agar containing a suspension of thepathogen of interest. Following incubation, clear inhibition zonesdevelop around the discs that contain an effective concentration of theantipathogenic polypeptide (Liu et al. (1994) Proc. Natl. Acad. Sci. USA91:1888-1892, herein incorporated by reference). Additionally,microspectrophotometrical analysis can be used to measure the in vitroantipathogenic properties of a composition (Hu et al. (1997) Plant Mol.Biol. 34:949-959 and Cammue et al. (1992) J. Biol. Chem. 267: 2228-2233,both of which are herein incorporated by reference).

Compositions and methods for controlling pathogenic agents are providedin the embodiments. The anti-pathogenic compositions comprise plantcyclotide nucleotide and amino acid sequences. Particularly, the plantnucleic acid and amino acid sequences and fragments and variants thereofset forth herein possess anti-pathogenic activity. Accordingly, thecompositions and methods are useful in protecting plants against fungalpathogens, viruses, nematodes, and the like. Additionally provided aretransformed plants, plant cells, plant tissues and seeds thereof.

The compositions of the embodiments can be used in a variety of methodswhereby the protein products can be expressed in crop plants to functionas antimicrobial proteins. The compositions of the embodiments may beexpressed in a crop plant such as maize or soybean to function as anantifungal agent, an antinematodal agent, and the like. Expression ofthe proteins of the embodiments will result in alterations or modulationof the level, tissue, or timing of expression to achieve enhanceddisease, nematode, viral, or fungal resistance.

The coding sequence for the cyclotide can be used in combination with apromoter that is introduced into a crop plant. In one embodiment, ahigh-level expressing constitutive promoter may be utilized and wouldresult in high levels of expression of the cyclotide. In otherembodiments, the coding sequence may be operably linked to atissue-preferred promoter to direct the expression to a plant tissueknown to be susceptible to a pathogen. Likewise, manipulation of thetiming of expression may be utilized. For example, by judicious choiceof promoter, expression can be enhanced early in plant growth to primethe plant to be responsive to pathogen attack. Likewise, pathogeninducible promoters can be used wherein expression of the cyclotide isturned on in the presence of the pathogen. If desired, a transit peptidecan be utilized to direct cellular localization of the protein product.In this manner, the native transit peptide or a heterologous transitpeptide can be used. However, it is recognized that both extracellularexpression and intracellular expression are encompassed by the methodsof the embodiments.

Sequences of the embodiments, as discussed in more detail below,encompass coding sequences, antisense sequences, and fragments andvariants thereof. Expression of the sequences of the embodiments can beused to modulate or regulate the expression of corresponding cyclotideproteins.

The compositions and methods of the embodiments can be used forenhancing resistance to plant pathogens including fungal pathogens,plant viruses, bacterial pathogens, nematodes, and the like. The methodinvolves stably transforming a plant with a nucleotide sequence capableof modulating the plant pathogen defense system operably linked with apromoter capable of driving expression of a gene in a plant cell. Asused herein, “enhancing resistance” is intended to mean increasing thetolerance of the plant to pathogens. That is, the cyclotide may slow orprevent pathogen infection and/or spread.

The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, “nucleic acid fragment”, and “isolated nucleic acid fragment”are used interchangeably herein. These terms encompass nucleotidesequences and the like. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof.

The term “isolated” polynucleotide refers to a polynucleotide that issubstantially free from other nucleic acid sequences, such as otherchromosomal and extrachromosomal DNA and RNA, that normally accompany orinteract with it as found in its naturally occurring environment.Isolated polynucleotides may be purified from a host cell in which theynaturally occur. Conventional nucleic acid purification methods known toskilled artisans may be used to obtain isolated polynucleotides. Theterm also embraces recombinant polynucleotides and chemicallysynthesized polynucleotides.

The term “recombinant” means, for example, that a nucleic acid sequenceis made by an artificial combination of two otherwise separated segmentsof sequence, e.g., by chemical synthesis or by the manipulation ofisolated nucleic acids by genetic engineering techniques.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough, for example, antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the embodiments such as deletion or insertion of one ormore nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theembodiments encompass more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof. Theterms “substantially similar” and “corresponding substantially” are usedinterchangeably herein.

Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the embodiments, whereinone or more nucleotides are substituted, deleted and/or inserted, fortheir ability to affect the level of the polypeptide encoded by theunmodified nucleic acid fragment in a plant or plant cell. For example,a substantially similar nucleic acid fragment representing at least oneof 30 contiguous nucleotides derived from the instant nucleic acidfragment can be constructed and introduced into a plant or plant cell.The level of the polypeptide encoded by the unmodified nucleic acidfragment present in a plant or plant cell exposed to the substantiallysimilar nucleic acid fragment can then be compared to the level of thepolypeptide in a plant or plant cell that is not exposed to thesubstantially similar nucleic acid fragment.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% sequence identity withthe gene to be suppressed. Moreover, alterations in a nucleic acidfragment that result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts.

Consequently, an isolated polynucleotide comprising a nucleotidesequence of at least one of 60 (preferably at least one of 40, mostpreferably at least one of 30) contiguous nucleotides derived from anucleotide sequence selected from the group consisting of SEQ ID NOs: 1and 3, and the complement of such nucleotide sequences may be used inmethods of selecting an isolated polynucleotide that affects theexpression of a plant cyclotide polypeptide in a host cell. A method ofselecting an isolated polynucleotide that affects the level ofexpression of a polypeptide in a virus or in a host cell (eukaryotic,such as plant or yeast, prokaryotic such as bacterial) may comprise thesteps of: constructing an isolated polynucleotide of the embodiments oran isolated chimeric gene of the embodiments; introducing the isolatedpolynucleotide or chimeric gene into a host cell; measuring the level ofa polypeptide or enzyme activity in the host cell containing theisolated polynucleotide; and comparing the level of a polypeptide orenzyme activity in the host cell containing the isolated polynucleotidewith the level of a polypeptide or enzyme activity in a host cell thatdoes not contain the isolated polynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms.

Specificity in hybridization is typically the function ofpost-hybridization washes, the critical factors being the ionic strengthand temperature of the final wash solution. The thermal melting point(T_(m)) is the temperature (under defined ionic strength and pH) atwhich 50% of a complementary target sequence hybridizes to a perfectlymatched probe. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. T_(m) is reduced by about 1° C. for each 1% ofmismatching; thus, T_(m), hybridization, and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with >90% identity are sought, the T_(m) can be decreased10° C. Generally, stringent conditions are selected to be about 5° C.lower than the T_(m) for the specific sequence and its complement at adefined ionic strength and pH. However, severely stringent conditionscan utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower thanthe T_(m); moderately stringent conditions can utilize a hybridizationand/or wash at 6, 7, 8, 9, or 10° C. lower than the T_(m); lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15, or 20° C. lower than the T_(m).

Using the equation, hybridization and wash compositions, and desiredT_(m), those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis preferred to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York). Also see Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.).

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl, 0.3 M trisodium citrate)at 50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C. for at least 4 hours, more preferably up to 12 hours or longer,and a final wash in 0.1×SSC at 60 to 65° C. for at least 20 minutes.Optionally, wash buffers may comprise about 1% SDS. Duration ofhybridization is generally less than about 24 hours, usually about 4 toabout 12 hours.

Thus, isolated sequences that encode a cyclotide polypeptide and whichhybridize under stringent conditions to the cyclotide sequencesdisclosed herein, or to fragments thereof, are encompassed by theembodiments.

Substantially similar nucleic acid fragments of the embodiments may alsobe characterized by the percent identity of the amino acid sequencesthat they encode. For example, isolated nucleic acids which encode apolypeptide with a given percent sequence identity to the polypeptidesof SEQ ID NO: 2 and 4 are disclosed. Identity can be calculated using,for example, the BLAST, CLUSTALW or GAP algorithms under defaultconditions. The percentage of identity to a reference sequence is atleast 50% and, rounded upwards to the nearest integer, can be expressedas an integer selected from the group of integers consisting of from 50to 99. Thus, for example, the percentage of identity to a referencesequence can be at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99%.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thelocal alignment method of Pearson and Lipman (1988) Proc. Natl. Acad.Sci. USA 85:2444-2448; the algorithm of Karlin and Altschul (1990) Proc.Natl. Acad. Sci. USA 87:2264, as modified in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0); the ALIGN PLUS program (Version 3.0,copyright 1997); and GAP, BESTFIT, BLAST, FASTA, and TFASTA in theWisconsin Genetics Software Package of Genetics Computer Group, Version10 (available from Accelrys, 9685 Scranton Road, San Diego, Calif.,92121, USA). The scoring matrix used in Version 10 of the WisconsinGenetics Software Package is BLOSUM62 (see Henikoff and Henikoff (1989)Proc. Natl. Acad. Sci. USA 89:10915). Alignments using these programscan be performed using the default parameters. As used herein “defaultvalues” will mean any set of values or parameters which originally loadwith the software when first initialized.

The GAP program uses the algorithm of Needleman and Wunsch (1970) supra,to find the alignment of two complete sequences that maximizes thenumber of matches and minimizes the number of gaps. GAP considers allpossible alignments and gap positions and creates the alignment with thelargest number of matched bases and the fewest gaps. It allows for theprovision of a gap creation penalty and a gap extension penalty in unitsof matched bases. Default gap creation penalty values and gap extensionpenalty values in Version 10 of the Wisconsin Genetics Software Packagefor protein sequences are 8 and 2, respectively. For nucleotidesequences the default gap creation penalty is 50 while the default gapextension penalty is 3. The gap creation and gap extension penalties canbe expressed as an integer selected from the group of integersconsisting of from 0 to 200. Thus, for example, the gap creation and gapextension penalties can each be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

The CLUSTAL program is well described by Higgins et al. (1988) Gene73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al.(1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. TheALIGN and the ALIGN PLUS programs are based on the algorithm of Myersand Miller (1988) supra.

The BLAST (Basic Local Alignment Search Tool) programs of Altschul etal. (1993) J. Mol. Biol. 215:403-410 are based on the algorithm ofKarlin and Altschul (1990) supra. BLAST nucleotide searches can beperformed with the BLASTN program, which searches a nucleotide queryagainst a nucleotide database, to obtain nucleotide sequences homologousto a nucleotide sequence encoding a protein of the embodiments. BLASTprotein searches can be performed with the BLASTX program, whichsearches a nucleotide query against a peptide database, to obtain aminoacid sequences homologous to a protein or polypeptide of theembodiments. The TBLASTN program provides for a peptide query against anucleotide database, while the TBLASTX program allows for a nucleotidequery against a nucleotide database with the translation of both toprotein. To obtain gapped alignments for comparison purposes, GappedBLAST (in BLAST 2.0) can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST2.0) can be used to perform an iterated search that detects distantrelationships between molecules (see Altschul et al. (1997) supra). Whenutilizing any BLAST program the default parameters of the respectiveprograms can be used. Alignment may also be performed manually byinspection.

An “equivalent program” refers to any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by the preferred program.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences makes reference to the residues inthe two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins, it is recognizedthat residue positions which are not identical often differ byconservative amino acid substitutions where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. When sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of one, and a non-conservative substitution is given a score ofzero, a conservative substitution is given a score between zero and one.The scoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence for optimal alignment of the twosequences. The percentage may be calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison, and multiplying the result by 100to yield the percentage of sequence identity.

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

As used herein, “comparison window” makes reference to a contiguous andspecified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e, gaps) compared to the reference sequence for optimalalignment of the two sequences. Generally, the comparison window is atleast 20 contiguous nucleotides in length, and optionally can be 30, 40,50, 100, or longer. Those of skill in the art understand that to avoid ahigh similarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence, a gap penalty is typically introduced and issubtracted from the number of matches.

As used herein, “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire nucleicacid sequence or the entire amino acid sequence of a native(non-synthetic), endogenous sequence. A full-length polynucleotideencodes the full-length form of the specified protein.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 70% sequenceidentity, for example, at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identitycompared to a reference sequence using one of the alignment programsdescribed using standard parameters. One of skill in the art willrecognize that these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning, and the like. Substantial identity of amino acidsequences for these purposes normally means sequence identity of atleast 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, and 99%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the T_(m) for the specific sequence at a defined ionic strength andpH. However, stringent conditions encompass temperatures in the range ofabout 1° C. to about 20° C., depending upon the desired degree ofstringency as otherwise qualified herein. Nucleic acids that do nothybridize to each other under stringent conditions are stillsubstantially identical if the polypeptides they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is when the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

The term “substantial identity” in the context of a peptide indicatesthat a peptide comprises a sequence with at least 70% sequence identityto a reference sequence, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to the reference sequenceover a specified comparison window. Preferably, optimal alignment isconducted using the homology alignment algorithm of Needleman and Wunsch(1970) supra. An indication that two peptide sequences are substantiallyidentical is that one peptide is immunologically reactive withantibodies raised against the second peptide. Thus, a peptide issubstantially identical to a second peptide, for example, where the twopeptides differ only by a conservative substitution. Peptides that are“substantially similar” share sequences as noted above except thatresidue positions that are not identical may differ by conservativeamino acid changes.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as the BLAST programs discussed elsewhere inthis specification. (Altschul et al. (1993) supra).

Accordingly, a “substantial portion” of a nucleotide sequence comprisesa nucleotide sequence that will afford specific identification and/orisolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the embodiments comprise the complete sequences as reportedin the accompanying Sequence Listing, as well as substantial portions ofthose sequences as defined above.

Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are also encompassed by the embodiments. A“fragment” is a portion of the nucleotide sequence or a portion of theamino acid sequence, and hence protein, encoded thereby. The nucleicacid fragments of the embodiments may be used to isolate cDNAs and genesencoding homologous proteins from the same or other plant species.

Isolation of homologous genes using sequence-dependent protocols is wellknown in the art. Examples of sequence-dependent protocols include, butare not limited to, methods of nucleic acid hybridization, and methodsof DNA and RNA amplification as exemplified by various uses of nucleicacid amplification technologies. “PCR” or “polymerase chain reaction” isa technique used for the amplification of specific DNA segments (U.S.Pat. Nos. 4,683,195 and 4,800,159).

Genes encoding other plant cyclotides, either as cDNAs or genomic DNAs,could be isolated directly by using all or a portion of the instantnucleic acid fragments as DNA hybridization probes to screen librariesfrom any desired plant employing methodology well known to those skilledin the art. Specific oligonucleotide probes based upon the instantnucleic acid sequences can be designed and synthesized by methods knownin the art (Sambrook et al. (1989), supra). Moreover, the entiresequences can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primer DNA labeling, nicktranslation, or end-labeling techniques, or RNA probes using availablein vitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part or all of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).Consequently, a polynucleotide comprising a nucleotide sequence of atleast one of 60 (preferably one of at least 40, most preferably one ofat least 30) contiguous nucleotides derived from a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 1 or 3, and thecomplement of such nucleotide sequences may be used in such methods toobtain a nucleic acid fragment encoding a substantial portion of anamino acid sequence of a polypeptide of the embodiments.

The embodiments relate to a method of obtaining a nucleic acid fragmentencoding a substantial portion of a cyclotide polypeptide comprising thesteps of: synthesizing an oligonucleotide primer comprising a nucleotidesequence of at least one of 60, preferably at least one of 40, mostpreferably at least one of 30 contiguous nucleotides derived from anucleotide sequence selected from the group consisting of SEQ ID NOs:1or 3, and the complement of such nucleotide sequences; and amplifying anucleic acid fragment using the oligonucleotide primer. The amplifiednucleic acid fragment preferably will encode a portion of a plantcyclotide polypeptide.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1-34; Sambrook et al. (1989) supra).

Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the native protein and hence havecyclotide-like activity and thereby affect responses to pathogens.Alternatively, fragments of a nucleotide sequence that are useful ashybridization probes generally do not encode protein fragments retainingbiological activity. Thus, fragments of a nucleotide sequence may rangefrom at least about 20 nucleotides, about 50 nucleotides, about 100nucleotides, and up to the full-length nucleotide sequence encoding theproteins of the embodiments.

A fragment of a cyclotide nucleotide sequence that encodes abiologically active portion of a cyclotide protein of the embodimentswill encode at least 10, 15, 25, 30, 50, 100, contiguous amino acids, orup to the total number of amino acids present in a full-length proteinof the embodiments. Fragments of a cyclotide nucleotide sequence thatare useful as hybridization probes for PCR primers generally need notencode a biologically active portion of a cyclotide protein.

Thus, a fragment of a cyclotide nucleotide sequence may encode abiologically active portion of a cyclotide protein, or it may be afragment that can be used as a hybridization probe or PCR primer usingmethods disclosed herein. A biologically active portion of a cyclotideprotein can be prepared by isolating a portion of one of the cyclotidenucleotide sequences of the embodiments, expressing the encoded portionof the cyclotide protein (e.g., by recombinant expression in vitro), andassessing the activity of the encoded portion of the cyclotide protein.Nucleic acid molecules that are fragments of a cyclotide nucleotidesequence comprise at least 16, 20, 30, 40, 50, 75, 100, 150, 200, 250,or 300 nucleotides, or up to the number of nucleotides present in afull-length cyclotide nucleotide sequence disclosed herein.

The biological activity of the cyclotide polypeptides affecting theplant defense response can be assayed by any method known in the art(see for example, U.S. Pat. No. 5,614,395; Thomma et al. (1998) Proc.Natl. Acad. Sci. USA 95:15107-5111; Liu et al. (1994) supra; Hu et al.(1997) supra; Cammue et al. (1992) supra; and Thevissen et al. (1996) J.Biol. Chem. 271:15018-15025, all of which are herein incorporated byreference). Furthermore, assays to detect cyclotide-like activityinclude, for example, assessing antifungal and/or antimicrobial activity(see Terras et al. (1992) J. Biol. Chem. 267:15301-15309; Terras et al.(1993) Plant Physiol (Bethesda) 103:1311-1319; Terras et al. (1995)Plant Cell 7:573-588, Moreno et al. (1994) Eur. J. Biochem. 223:135-139;and Osborn et al. (1995) FEBS Lett. 368:257-262, all of which are hereinincorporated by reference).

The term “variants” is used to mean substantially similar. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of one of the cyclotide polypeptides of the embodiments.Naturally occurring allelic variants such as these can be identifiedwith the use of well-known molecular biology techniques, such as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined herein. Variant nucleotide sequences also includesynthetically derived nucleotide sequences, such as those generated, forexample, by using site-directed mutagenesis but which still encode acyclotide protein of the embodiments. Generally, variants of aparticular nucleotide sequence of the embodiments will have at leastabout 50%, 60%, 65%, 70%, generally at least about 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to that particular nucleotidesequence as determined by sequence alignment programs describedelsewhere herein using default parameters.

The term “variant protein” is intended to mean a protein derived fromthe native protein by deletion (so-called truncation) or addition of oneor more amino acids to the N-terminal and/or C-terminal end of thenative protein; deletion or addition of one or more amino acids at oneor more sites in the native protein; or substitution of one or moreamino acids at one or more sites in the native protein. Variant proteinsencompassed by the present embodiments are biologically active, that isthey continue to possess the desired biological activity of the nativeprotein, that is, cyclotide-like activity as described herein. Suchvariants may result from, for example, genetic polymorphism or fromhuman manipulation. Biologically active variants of a native cyclotideprotein of the embodiments will have at least about 40%, 50%, 60%, 65%,70%, generally at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acidsequence for the native protein as determined by sequence alignmentprograms described elsewhere herein using default parameters. Abiologically active variant of a protein of the embodiments may differfrom that protein by as few as 1-15 amino acid residues, as few as 1-10,such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acidresidue.

The polypeptides of the embodiments may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Novel proteins having properties of interest may be createdby combining elements and fragments of proteins of the embodiments withother proteins as well. Methods for such manipulations are generallyknown in the art. For example, amino acid sequence variants of thecyclotide proteins can be prepared by mutations in the DNA. Methods formutagenesis and nucleotide sequence alterations are well known in theart. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S.Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (Macmillan Publishing Company, New York) and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be preferred.

Thus, the genes and nucleotide sequences of the embodiments include bothnaturally occurring sequences as well as mutant forms. Likewise, theproteins of the embodiments encompass naturally occurring proteins aswell as variations and modified forms thereof. Such variants willcontinue to possess the desired cyclotide-like activity or defenseresponse activity. Obviously, mutations that will be made in the DNAencoding the variant must not place the sequence out of reading frameand preferably will not create complementary regions that could producesecondary mRNA structure (see EP Patent Publication No. 0 075 444 B1).

In nature, some polypeptides are produced as complex precursors which,in addition to targeting labels such as the signal peptides discussedelsewhere in this application, also contain other fragments of peptideswhich are removed (processed) at some point during protein maturation,resulting in a mature form of the polypeptide that is different from theprimary translation product. “Mature protein” refers to apost-translationally processed polypeptide; i.e., one from which anypre- or propeptides present in the primary translation product have beenremoved. “Precursor protein” or “prepropeptide” or “preproprotein” allrefer to the primary product of translation of mRNA; i.e., with pre- andpropeptides still present. Pre- and propeptides may include, but are notlimited to, intracellular localization signals. The form of thetranslation product with only the signal peptide removed but no furtherprocessing yet is called a “propeptide” or “proprotein”. The fragmentsor segments to be removed may themselves also be referred to as“propeptides.” A proprotein or propeptide thus has had the signalpeptide removed, but contains propeptides and the portions that willmake up the mature protein. The skilled artisan is able to determine,depending on the species in which the proteins are being expressed andthe desired intracellular location, if higher expression levels might beobtained by using a gene construct encoding just the mature form of theprotein, the mature form with a signal peptide, or the proprotein (i.e.,a form including propeptides) with a signal peptide. For optimalfunctional expression in plants, the pre- and propeptide sequences maybe needed. The propeptide segments may play a role in aiding correctpeptide folding, or in the case of cyclotides, may aid in correctcyclization.

SEQ ID NO: 1 is a viola sequence encoding the full length preproproteinof SEQ ID NO: 2. This preproprotein is further processed in the cell toproduce the cyclizable form of the cyclotide, SEQ ID NO: 6. SEQ ID NO: 3is a viola sequence encoding the full length preproprotein of SEQ ID NO:4. This preproprotein is further processed in the cell to produce thecyclizable form of the cyclotide, SEQ ID NO: 7.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. Biological activity ofpolypeptides (i.e., influencing the plant defense response and variousdevelopmental pathways, including, for example, influencing celldivision) can be assayed by any method known in the art. Biologicalactivity of the variant polypeptides of the embodiments can be assayedby any method known in the art, such as those already discussed andreferenced elsewhere in this application.

Variant nucleotide sequences and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different cyclotidecoding sequences can be manipulated to create a new cyclotide proteinpossessing the desired properties. In this manner, libraries ofrecombinant polynucleotides are generated from a population of relatedsequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. Strategies for such DNA shuffling are known in theart. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997)Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. Accordingly, the embodiments relateto any nucleic acid fragment comprising a nucleotide sequence thatencodes all or a substantial portion of the amino acid sequences setforth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.Determination of preferred codons can be based on a survey of genesderived from the host cell where sequence information is available. Forexample, the codon frequency tables available on the world wide web atKazusa.or.jp/codon/ may be used to determine preferred codons for avariety of organisms. See also Campbell and Gowri (1990) Plant Physiol.92:1-11; Murray et al. (1989) Nucleic Acids Res. 17:477-498, U.S. Pat.Nos. 5,380,831 and 5,436,391; and the information found on the worldwide web at agron.missouri.edu/mnl/77/10simmons.html; hereinincorporated by reference.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to a nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments which are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of thenucleotide sequence to reflect the codon bias of the host cell. Theskilled artisan appreciates the likelihood of successful gene expressionif codon usage is biased towards those codons favored by the host.Determination of preferred codons can be based on a survey of genesderived from the host cell where sequence information is available.

It is to be understood that as used herein the term “transgenic”includes any cell, cell line, callus, tissue, plant part, or plant thegenotype of which has been altered by the presence of a heterologousnucleic acid including those transgenics initially so altered as well asthose created by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation.

A transgenic “event” is produced by transformation of plant cells with aheterologous DNA construct, including a nucleic acid expression cassettethat comprises a transgene of interest, the regeneration of a populationof plants resulting from the insertion of the transgene into the genomeof the plant, and selection of a particular plant characterized byinsertion into a particular genome location. An event is characterizedphenotypically by the expression of the transgene. At the genetic level,an event is part of the genetic makeup of a plant. The term “event” alsorefers to progeny produced by a sexual outcross between the transformantand another variety that include the heterologous DNA.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, andprogeny of same. Parts of transgenic plants are to be understood to bewithin the scope of the embodiments and to comprise, for example, plantcells, protoplasts, tissues, callus, embryos, as well as flowers,ovules, stems, fruits, leaves, roots originating in transgenic plants ortheir progeny previously transformed with a DNA molecule of theembodiments and therefore consisting at least in part of transgeniccells.

As used herein, the term “plant cell” includes, without limitation,seeds, suspension cultures, embryos, meristematic regions, callustissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores. The class of plants that can be used in the methods of theembodiments is generally as broad as the class of higher plants amenableto transformation techniques, including both monocotyledonous anddicotyledonous plants.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. As used herein, the terms “encoding” or“encoded” when used in the context of a specified nucleic acid mean thatthe nucleic acid comprises the requisite information to guidetranslation of the nucleotide sequence into a specified protein. Theinformation by which a protein is encoded is specified by the use ofcodons. A nucleic acid encoding a protein may comprise non-translatedsequences (e.g., introns) within translated regions of the nucleic acidor may lack such intervening non-translated sequences (e.g., as incDNA).

“Regulatory sequences” refer to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, and polyadenylation recognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequence mayconsist of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence that can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters that cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. While new promoters of various types useful in plant cellsare constantly being discovered; numerous examples of known promotersmay be found, for example, in the compilation by Okamuro and Goldberg(1989) Biochemistry of Plants 15:1-82. It is further recognized thatsince in most cases the exact boundaries of regulatory sequences havenot been completely defined, nucleic acid fragments of different lengthsmay have identical promoter activity.

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

The “5′ leader sequence,” “5′ non-coding sequence,” or “translationleader sequence” refers to a nucleotide sequence located between thepromoter sequence of a gene and the coding sequence. The translationleader sequence is present in the fully processed mRNA upstream of thetranslation start sequence. The translation leader sequence may affectprocessing of the primary transcript to mRNA, mRNA stability ortranslation efficiency. Examples of translation leader sequences havebeen described (Turner and Foster (1995) Mol. Biotechnol. 3:225-236). A5′ non-translated leader sequence is usually characterized as thatportion of the mRNA molecule which most typically extends from the 5′CAP site to the AUG protein translation initiation codon.

Other methods known to enhance translation and/or mRNA stability canalso be utilized, for example, introns, such as the maize Ubiquitinintron (Christensen and Quail (1996) Transgenic Res. 5:213-218 andChristensen et al. (1992) Plant Molecular Biology 18:675-689) or themaize AdhI intron (Kyozuka et al. (1991) Mol. Gen. Genet. 228:40-48 andKyozuka et al. (1990) Maydica 35:353-357), and the like. Various intronsequences have been shown to enhance expression, particularly inmonocotyledonous cells. The introns of the maize AdhI gene have beenfound to significantly enhance the expression of the wild-type geneunder its cognate promoter when introduced into maize cells. Intron 1was found to be particularly effective and enhanced expression in fusionconstructs with the chloramphenicol acetyltransferase gene (Callis etal., (1987) Genes Develop. 1:1183-1200). In the same experimentalsystem, the intron from the maize bronzel gene had a similar effect inenhancing expression. The AdhI intron has also been shown to enhance CATexpression 12-fold (Mascarenhas et al. (1990) Plant Mol. Biol.6:913-920). Intron sequences have routinely been incorporated into planttransformation vectors, typically within the non-translated leader.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be an RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptides by the cell. “cDNA” refers to a DNA that is complementaryto and derived from an mRNA template. The cDNA can be single-stranded orconverted to double stranded form using, for example, the Klenowfragment of DNA polymerase 1. “Sense” RNA refers to an RNA transcriptthat includes the mRNA and so can be translated into a polypeptide bythe cell. “Antisense”, when used in the context of a particularnucleotide sequence, refers to the complementary strand of the referencetranscription product. “Antisense RNA” refers to an RNA transcript thatis complementary to all or part of a target primary transcript or mRNAand that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous nucleotidesequence can be from a species different from that from which thenucleotide sequence was derived, or, if from the same species, thepromoter is not naturally found operably linked to the nucleotidesequence. A heterologous protein may originate from a foreign species,or, if from the same species, is substantially modified from itsoriginal form by deliberate human intervention.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the embodiments. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Underexpression”refers to the production of a gene product in transgenic organisms atlevels below that of levels of production in normal or non-transformedorganisms. “Co-suppression” refers to the production of sense RNAtranscripts capable of suppressing the expression of identical orsubstantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference).

A “protein” or “polypeptide” is a chain of amino acids arranged in aspecific order determined by the coding sequence in a polynucleotideencoding the polypeptide.

“Altered levels” or “altered expression” refers to the production ofgene product(s) in transgenic organisms in amounts or proportions thatdiffer from that of normal or non-transformed organisms.

“Null mutant” refers here to a host cell that either lacks theexpression of a certain polypeptide or expresses a polypeptide which isinactive or does not have any detectable expected enzymatic function.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product has been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be, but are not limited to,intracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence that istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence that is translated in conjunction with aprotein and directs the protein to the secretory system (see Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal can furtherbe added, or if to the endoplasmic reticulum, an endoplasmic reticulumretention signal may be added. If the protein is to be directed to thenucleus, any signal peptide present should be removed and instead anuclear localization signal included (see Raikhel (1992) Plant Phys.100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Additional transformation methods aredisclosed below. Thus, isolated polynucleotides of the embodiments canbe incorporated into recombinant constructs, typically DNA constructs,capable of introduction into and replication in a host cell. Such aconstruct can be a vector that includes a replication system andsequences that are capable of transcription and translation of apolypeptide-encoding sequence in a given host cell. Vectors suitable forstable transfection of plant cells or for the establishment oftransgenic plants have been described in, e.g., Pouwels et al., (1985;Supp. 1987) Cloning Vectors: A Laboratory Manual, Weissbach andWeissbach (1989) Methods for Plant Molecular Biology, (Academic Press,New York); and Flevin et al., (1990) Plant Molecular Biology Manual,(Kluwer Academic Publishers). Typically, plant expression vectorsinclude, for example, one or more cloned plant genes under thetranscriptional control of 5′ and 3′ regulatory sequences and a dominantselectable marker. Such plant expression vectors also can contain apromoter regulatory region (e.g., a regulatory region controllinginducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific expression), atranscription initiation start site, a ribosome binding site, an RNAprocessing signal, a transcription termination site, and/or apolyadenylation signal.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. (1989) supra.

Another embodiment concerns viruses and host cells comprising either thechimeric genes of the embodiments as described herein or an isolatedpolynucleotide of the embodiments as described herein. Examples of hostcells that can be used to practice the embodiments include, but are notlimited to, yeast, bacterial, fungal, insect, amphibian, mammalian, andplant cells.

As used herein, “host cell” refers to a cell which comprises aheterologous nucleic acid sequence of the embodiments. Host cells may beprokaryotic cells such as E. coli, or eukaryotic cells such as yeast,fungal, insect, amphibian, mammalian or plant cells. Preferably, hostcells are monocotyledonous or dicotyledonous plant cells. A particularlypreferred dicotyledonous host cell is a soybean host cell.

Overexpression of the proteins of the embodiments may be accomplished byfirst constructing a chimeric gene in which the coding region isoperably linked to a promoter capable of directing expression of a genein the desired tissues at the desired stage of development. The chimericgene may comprise promoter sequences and translation leader sequencesderived from the same genes. 3′ non-coding sequences encodingtranscription termination signals may also be provided. The instantchimeric gene may also comprise one or more introns in order tofacilitate gene expression.

The cyclotide sequences of the embodiments are provided in expressioncassettes or DNA constructs for expression in the plant of interest. Thecassette will include 5′ and 3′ regulatory sequences operably linked toa cyclotide sequence of the embodiments. The cassette may additionallycontain at least one additional gene to be cotransformed into theorganism. Alternatively, the additional gene(s) can be provided onmultiple expression cassettes.

Such an expression cassette is provided with a plurality of restrictionsites for insertion of the cyclotide sequence to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

The expression cassette will include, in the 5′-3′ direction oftranscription, a transcriptional initiation region (i.e., a promoter),translational initiation region, a polynucleotide of the invention, atranslational termination region and, optionally, a transcriptionaltermination region functional in the host organism. The regulatoryregions (i.e., promoters, transcriptional regulatory regions, andtranslational termination regions) and/or the polynucleotide of theinvention may be native/analogous to the host cell or to each other.Alternatively, the regulatory regions and/or the polynucleotide of theinvention may be heterologous to the host cell or to each other. As usedherein, “heterologous” in reference to a sequence is a sequence thatoriginates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus, or the promoteris not the native promoter for the operably linked polynucleotide.

The optionally included termination region may be native with thetranscriptional initiation region, may be native with the operablylinked DNA sequence of interest, or may be derived from another source.Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthase (NOS)termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991)Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroeet al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Additional sequence modifications are known to enhance gene expressionin a cellular host, including elimination of sequences encoding spuriouspolyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238); MDMVleader (Maize Dwarf Mosaic Virus), and human immunoglobulin heavy-chainbinding protein (BiP) (Macejak et al. (1991) Nature 353:90-94);untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaicvirus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA,ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottlevirus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385). Seealso, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. Othermethods known to enhance translation can also be utilized, for example,introns, and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. To thisend, adapters or linkers may be employed to join the DNA fragments orother manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glyphosate, glufosinate ammonium, bromoxynil, imidazolinones,and 2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992)Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl.Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff(1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon,pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989)Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerst et al. (1989) Proc.Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg;Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow etal. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc.Natl. Acad. Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad.Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res.19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol.10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother.35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104;Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992)Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbookof Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill etal. (1988) Nature 334:721-724, and copending U.S. patent applicationSer. Nos. 10/004,357 and 10/427,692. Such disclosures are hereinincorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the embodiments.

In specific embodiments, methods for increasing pathogen resistance in aplant comprise stably transforming a plant with a DNA constructcomprising an antipathogenic nucleotide sequence of the embodimentsoperably linked to a promoter that drives expression in a plant. Suchmethods find use in agriculture particularly in limiting the impact ofplant pathogens on crop plants. While the choice of promoter will dependon the desired timing and location of expression of the anti-pathogenicnucleotide sequences, preferred promoters include constitutive andpathogen-inducible promoters.

A number of promoters can be used in the practice of the embodiments.The promoters can be selected based on the desired outcome. That is, thenucleic acids can be combined with constitutive, tissue-preferred, orother promoters for expression in the host cell of interest. Suchconstitutive promoters include, for example, the core promoter of theRsyn7 promoter and other constitutive promoters disclosed in WO 99/43838and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al.(1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689);PEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten etal. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026),and the like. Other constitutive promoters include, for example, thosedisclosed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611, hereinincorporated by reference.

Generally, it will be beneficial to express the gene from an induciblepromoter, for example from a pathogen-inducible promoter. Such promotersinclude those from pathogenesis-related proteins (PR proteins), whichare induced following infection by a pathogen; e.g., PR proteins, SARproteins, beta-1,3-glucanase, chitinase, etc. See, for example, Redolfiet al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992)Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116.See also WO 99/43819 published Sep. 9, 1999, herein incorporated byreference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau et al. (1987) PlantMol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; andYang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen etal. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad.Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertzet al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible); and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero et al. (1992) Physiol. Mol. Plant. Path. 41:189-200).

Additionally, as pathogens find entry into plants through wounds orinsect damage, a wound-inducible promoter may be used in theconstructions of the embodiments. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like,herein incorporated by reference.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters. See, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156; herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced cyclotideexpression within a particular plant tissue. Tissue-preferred promotersinclude Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al.(1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen.Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res.6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341;Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al.(1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant CellPhysiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ.20:181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138;Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; andGuevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters canbe modified, if necessary, for weak expression.

Leaf-specific promoters are known in the art. See, for example, Yamamotoet al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol.105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778;Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol.Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci.USA 90(20): 9586-9590.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphatesynthase); and celA (cellulose synthase) (see WO 00/11177, hereinincorporated by reference). Gama-zein is a preferred endosperm-specificpromoter. Glob-1 is a preferred embryo-specific promoter. For dicots,seed-specific promoters include, but are not limited to, beanβ-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and thelike. For monocots, seed-specific promoters include, but are not limitedto, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken1, shrunken 2, globulin 1, etc. See also WO 00/12733, whereseed-preferred promoters from end1 and end2 genes are disclosed; hereinincorporated by reference.

The method of transformation/transfection is not critical to theembodiments. Various methods of transformation or transfection arecurrently available. As newer methods are available to transform cropsor other host cells they may be used with the embodiments. Accordingly,a wide variety of methods have been developed to insert a DNA sequenceinto the genome of a host cell to obtain the transcription and/ortranslation of the sequence to effect phenotypic changes in theorganism. The nucleic acid fragments of the embodiments may be used tocreate transgenic plants in which the disclosed plant cyclotides arepresent at higher or lower levels than normal or in cell types ordevelopmental stages in which they are not normally found. This wouldhave the effect of altering the level of disease (e.g., fungal) andpathogen resistance in those cells. Thus, any method, which provides foreffective transformation/transfection may be employed.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J.3:2717-2722), and ballistic particle acceleration (see, for example,U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; and 5,932,782; McCabe etal. (1988) Biotechnology 6:923-926); and Lec1 transformation (WO00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet.22:421-477; Sanford et al. (1987) Particulate Science and Technology5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean);Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182(soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean);Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988)Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and5,324,646; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize);Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al.(1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al.(1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals);Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet et al. (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209(pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 andKaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediatedtransformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505(electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 andChristou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda etal. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacteriumtumefaciens); all of which are herein incorporated by reference.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensurethat expression of the desired phenotypic characteristic has beenachieved.

The sequences presented in the embodiments may be used fortransformation of any plant species, including, but not limited to,monocots and dicots. Examples of plants of interest include, but are notlimited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.juncea), particularly those Brassica species useful as sources of seedoil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum),foxtail millet (Setaria italica), finger millet (Eleusine coracana)),sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoeabatatas), cassaya (Manihot esculenta), coffee (Coffea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya(Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamiaintegrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris),sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, andconifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp., Pisum spp.), and members of the genusCucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis),and musk melon (C. melo). Ornamentals include azalea (Rhododendronspp.), hydrangea (Hydrangea macrophylla), hibiscus (Hibiscusrosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils(Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthuscaryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.Conifers that may be employed in practicing the embodiments include, forexample, pines such as loblolly pine (Pinus taeda), slash pine (Pinuselliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinuscontorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsugamenziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Piceaglauca); redwood (Sequoia sempervirens); true firs such as silver fir(Abies amabilis) and balsam fir (Abies balsamea); and cedars such asWestern red cedar (Thuja plicata) and Alaska yellow cedar (Chamaecyparisnootkatensis).

Prokaryotic cells may be used as hosts for expression. Prokaryotes mostfrequently are represented by various strains of E. coli; however, othermicrobial strains may also be used. Commonly used prokaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding sequences, include such commonly used promoters as thebeta lactamase (penicillinase) and lactose (lac) promoter systems (Changet al. (1977) Nature 198:1056), the tryptophan (trp) promoter system(Goeddel et al. (1980) Nucleic Acids Res. 8:4057) and the lambda derivedPL promoter and N-gene ribosome binding site (Simatake and Rosenberg(1981) Nature 292:128). Examples of selection markers for E. coliinclude, for example, genes specifying resistance to ampicillin,tetracycline, or chloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein of the embodiments areavailable using Bacillus sp. and Salmonella (Palva et al. (1983) Gene22:229-235 and Mosbach et al. (1983) Nature 302:543-545).

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known to those of skill in theart. As explained briefly below, a polynucleotide of the embodiments canbe expressed in these eukaryotic systems. In some embodiments,transformed/transfected plant cells, as discussed infra, are employed asexpression systems for production of the proteins of the embodiments.Such antimicrobial proteins can be used for any application includingcoating surfaces to target microbes as described further infra.

Synthesis of heterologous nucleotide sequences in yeast is well known.Sherman, et al. (1982) Methods in Yeast Genetics (Cold Spring HarborLaboratory) is a well recognized work describing the various methodsavailable to produce proteins in yeast. Two widely utilized yeasts forproduction of eukaryotic proteins are Saccharomyces cerevisiae andPichia pastoris. Vectors, strains, and protocols for expression inSaccharomyces and Pichia are known in the art and available fromcommercial suppliers (e.g., Invitrogen). Suitable vectors usually haveexpression control sequences, such as promoters, including3-phosphoglycerate kinase or alcohol oxidase, and an origin ofreplication, termination sequences and the like, as desired.

A protein of the embodiments, once expressed, can be isolated from yeastby lysing the cells and applying standard protein isolation techniquesto the lysates. The monitoring of the purification process can beaccomplished by using Western blot techniques, radioimmunoassay, orother standard immunoassay techniques.

The sequences of the embodiments can also be ligated to variousexpression vectors for use in transfecting cell cultures of, forinstance, mammalian, insect, or plant origin. Illustrative cell culturesuseful for the production of the peptides are mammalian cells. A numberof suitable host cell lines capable of expressing intact proteins havebeen developed in the art, and include the HEK293, BHK21, and CHO celllines. Expression vectors for these cells can include expression controlsequences, such as an origin of replication, a promoter (e.g. the CMVpromoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter),an enhancer (Queen et al. (1986) Immunol. Rev. 89:49), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites (e.g., an SV40 large T Ag poly A additionsite), and transcriptional terminator sequences. Other animal cellsuseful for production of proteins of the embodiments are available, forinstance, from the American Type Culture Collection.

Appropriate vectors for expressing proteins of the embodiments in insectcells are usually derived from the SF9 baculovirus. Suitable insect celllines include mosquito larvae, silkworm, armyworm, moth and Drosophilacell lines such as a Schneider cell line (See, Schneider (1987) J.Embryol. Exp. Morphol. 27:353-365).

As with yeast, when higher animal or plant host cells are employed,polyadenylation or transcription terminator sequences are typicallyincorporated into the vector. An example of a terminator sequence is thepolyadenylation sequence from the bovine growth hormone gene. Sequencesfor accurate splicing of the transcript may also be included. An exampleof a splicing sequence is the VP1 intron from SV40 (Sprague et al.(1983) J. Virol. 45:773-781). Additionally, gene sequences to controlreplication in the host cell may be incorporated into the vector such asthose found in bovine papilloma virus type-vectors. Saveria-Campo (1985)“Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector,” in DNA CloningVol. II: A Practical Approach, ed. D. M. Glover (IRL Press, Arlington,Va.), pp. 213-238.

Animal and lower eukaryotic (e.g., yeast) host cells are competent orrendered competent for transfection by various means. There are severalwell-known methods of introducing DNA into animal cells. These include:calcium phosphate precipitation, fusion of the recipient cells withbacterial protoplasts containing the DNA, treatment of the recipientcells with liposomes containing the DNA, DEAE dextrin, electroporation,biolistics, and micro-injection of the DNA directly into the cells. Thetransfected cells are cultured by means well known in the art. Kuchler(1997) Biochemical Methods in Cell Culture and Virology (Dowden,Hutchinson and Ross, Inc.).

Plasmid vectors comprising the instant isolated polynucleotide (orchimeric gene) may be constructed. The choice of plasmid vector isdependent upon the method that will be used to transform host plants.The skilled artisan is well aware of the genetic elements that must bepresent on the plasmid vector in order to successfully transform, selectand propagate host cells containing the chimeric gene. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen.Genetics 218:78-86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by directing the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) supra), or nuclearlocalization signals (Raikhel (1992) supra) with or without removingtargeting sequences that are already present. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of use may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression of aspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppression technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds. For example, one canscreen by looking for changes in gene expression by using antibodiesspecific for the protein encoded by the gene being suppressed, or onecould establish assays that specifically measure enzyme activity. Apreferred method will be one that allows large numbers of samples to beprocessed rapidly, since it will be expected that a large number oftransformants will be negative for the desired phenotype.

The embodiments also provide an isolated polypeptide selected from thegroup consisting of: a polypeptide comprising an amino acid sequence setforth in SEQ ID NO: 2 and 4; a polypeptide characterized by at least 97%identity to SEQ ID NO: 2 and 4; a polypeptide characterized by at least98% identity to SEQ ID NO: 2 and 4; and a polypeptide characterized byat least 99% identity to SEQ ID NO: 2 and 4.

The instant polypeptides are useful in methods for impacting a plantpathogen comprising introducing into a plant or cell thereof at leastone nucleotide construct comprising a nucleotide sequence of theembodiments operably linked to a promoter that drives expression of anoperably linked sequence in plant cells, wherein said nucleotidesequence is selected from the group consisting of: a nucleotide sequenceset forth in SEQ ID NO: 1 and 3; a nucleotide sequence that encodes apolypeptide having the amino acid sequence set forth in SEQ ID NO: 2 and4; a nucleotide sequence characterized by at least 85% sequence identityto the nucleotide sequence set forth in SEQ ID NO: 1 and 3; a nucleotidesequence characterized by at least 90% sequence identity to thenucleotide sequence set forth in SEQ ID NO: 1 and 3; a nucleotidesequence characterized by at least 95% sequence identity to thenucleotide sequence set forth in SEQ ID NO: 1 and 3; and a nucleotidesequence that comprises the complement of any one of the above.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the embodiments in situ in cells or invitro in cell extracts. Polyclonal cyclotide-like antibodies can beprepared by immunizing a suitable subject (e.g., rabbit, goat, mouse, orother mammal) with a cyclotide agent immunogen. The anti-cyclotideantibody titer in the immunized subject can be monitored over time bystandard techniques, such as with an enzyme linked immunosorbent assay(ELISA) using immobilized antimicrobial polypeptides. At an appropriatetime after immunization, e.g., when the anti-cyclotide agent antibodytiters are highest, antibody-producing cells can be obtained from thesubject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497, the human B cellhybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), theEBV-hybridoma technique (Cole et al. (1985) in Monoclonal Antibodies andCancer Therapy, ed. Reisfeld and Sell (Alan R. Liss, Inc., New York,N.Y.), pp. 77-96) or trioma techniques. The technology for producinghybridomas is well known (see generally Coligan et al., eds. (1994)Current Protocols in Immunology (John Wiley & Sons, Inc., New York,N.Y.); Galfre et al. (1977) Nature 266:55052; Kenneth (1980) inMonoclonal Antibodies: A New Dimension In Biological Analyses (PlenumPublishing Corp., New York); and Lerner (1981) Yale J. Biol. Med.54:387-402).

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-cyclotide-like antibody can be identified and isolatedby screening a recombinant combinatorial immunoglobulin library (e.g.,an antibody phage display library) with a cyclotide to thereby isolateimmunoglobulin library members that bind the defensive agent. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening an antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679;93/01288; WO 92/01047; 92/09690; and 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734. The antibodies can be used to identifyhomologs of the cyclotides of the embodiments.

All or a substantial portion of the polynucleotides of the embodimentsmay also be used as probes for genetically and physically mapping thegenes that they are a part of, and as markers for traits linked to thosegenes. Such information may be useful in plant breeding in order todevelop lines with desired phenotypes. For example, the instant nucleicacid fragments may be used as restriction fragment length polymorphism(RFLP) markers. Southern blots (Sambrook et al. (1989) supra) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the embodiments. The resulting banding patterns maythen be subjected to genetic analyses using computer programs such asMapMaker (Lander et al. (1987) Genomics 1:174-181) in order to constructa genetic map. In addition, the nucleic acid fragments of theembodiments may be used to probe Southern blots containing restrictionendonuclease-treated genomic DNAs of a set of individuals representingparent and progeny of a defined genetic cross. Segregation of the DNApolymorphisms is noted and used to calculate the position of the instantnucleic acid sequence in the genetic map previously obtained using thispopulation (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4:37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. in: Nonmammalian Genomic Analysis: APractical Guide, Academic Press, New York), 1996, pp. 319-346, andreferences cited therein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Res.5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci. USA 86:9402-9406; Koes et al.(1995) Proc. Natl. Acad. Sci. USA 92:8149-8153; Bensen et al. (1995)Plant Cell 7:75-84). The latter approach may be accomplished in twoways. First, short segments of the instant nucleic acid fragments may beused in polymerase chain reaction protocols in conjunction with amutation tag sequence primer on DNAs prepared from a population ofplants in which Mutator transposons or some other mutation-causing DNAelement has been introduced (see Bensen, supra). The amplification of aspecific DNA fragment with these primers indicates the insertion of themutation tag element in or near the plant gene encoding the instantpolypeptide. Alternatively, the instant nucleic acid fragment may beused as a hybridization probe against PCR amplification productsgenerated from the mutation population using the mutation tag sequenceprimer in conjunction with an arbitrary genomic site primer, such asthat for a restriction enzyme site-anchored synthetic adaptor. Witheither method, a plant containing a mutation in the endogenous geneencoding the instant polypeptide can be identified and obtained. Thismutant plant can then be used to determine or confirm the naturalfunction of the instant polypeptides disclosed herein.

It is understood in the art that plant DNA viruses and fungal pathogensremodel the control of the host replication and gene expressionmachinery to accomplish their own replication and effective infection.The embodiments may be useful in preventing such corruption of the cell.

The cyclotide sequences find use in disrupting cellular function ofplant pathogens as well as altering the defense mechanisms of a hostplant to enhance resistance to disease or other pathogens. While notwishing to be bound by any particular mechanism of action to enhancedisease resistance or pathogen resistance, the gene products of thecyclotide sequences function to inhibit or prevent diseases in a plant,or attack by plant pathogens.

The methods of the embodiments can be used with other methods availablein the art for enhancing disease and pathogen resistance in plants. Forexample, any one of a variety of second nucleotide sequences may beutilized, embodiments of the invention encompass those second nucleotidesequences that, when expressed in a plant, help to increase theresistance of a plant to pathogens. It is recognized that such secondnucleotide sequences may be used in either the sense or antisenseorientation depending on the desired outcome.

Pathogens of the embodiments include, but are not limited to, viruses orviroids, bacteria, nematodes, fungi, and the like. Viruses include anyplant virus, for example, tobacco or cucumber mosaic virus, ringspotvirus, necrosis virus, maize dwarf mosaic virus, etc. Specific fungaland viral pathogens for the major crops include, but are not limited tothe following: Soybeans: Phytophthora megasperma f.sp. glycinea,Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum,Fusarium spp., Diaporthe spp., Sclerotium rolfsii, Cercospora spp.,Peronospora manshurica, Colletotrichum dematium (Colletotrichumtruncatum), Corynespora cassiicola, Septoria glycines, Phyllostictasojicola, Alternaria alternata, Pseudomonas syringae p.v. glycinea,Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa, Phialophoragregata, Glomerella glycines, Phakopsora pachyrhizi, Pythium spp.,Soybean mosaic virus, Tobacco Ring spot virus, Tobacco Streak virus,Tomato spotted wilt virus; Canola: Albugo candida, Alternaria brassicae,Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum,Mycosphaerella brassiccola, Pythium ultimum, Peronospora parasitica,Fusarium roseum, Alternaria alternata; Alfalfa: Clavibacter Michigan'ssubsp. insidiosum, Pythium spp., Phytophthora megasperma, Peronosporatrifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis,Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium spp.,Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches, Stemphyliumspp.; Wheat: Pseudomonas spp., Urocystis agropyri, Xanthomonascampestris p.v. translucens, Alternaria alternata, Cladosporiumherbarum, Fusarium spp., Ustilago tritici, Ascochyta tritici,Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminisf.sp. tritici, Puccinia spp., Pyrenophora tritici-repentis, Septoriaspp., Pseudocercosporella herpotrichoides, Rhizoctonia spp.,Gaeumannomyces graminis var. tritici, Pythium spp., Bipolarissorokiniana, Claviceps purpurea, Tilletia spp., Barley Yellow DwarfVirus, Brome Mosaic Virus, Soil Borne Wheat Mosaic Virus, Wheat StreakMosaic Virus, Wheat Spindle Streak Virus, American Wheat Striate Virus,High Plains Virus, European wheat striate virus; Sunflower: Plasmophorahalstedii, Sclerotinia sclerotiorum, Aster Yellows, Septoria helianthi,Phomopsis helianthi, Alternaria spp., Botrytis cinerea, Phomamacdonaldii, Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopusspp., Puccinia helianthi, Verticillium dahliae, Erwinia carotovorum p.v.carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugotragopogonis; Corn: Fusarium spp., Erwinia spp., Gibberella zeae(Fusarium graminearum), Stenocarpella maydis (Diplodia maydis), Pythiumspp., Aspergillus flavus, Bipolaris maydis O, T (Cochliobolusheterostrophus), Helminthosporium carbonum I, II & III (Cochlioboluscarbonum), Exserohilum turcicum I, II & III, Helminthosporiumpedicellatum, Physoderma maydis, Phyllosticta maydis, Kabatiella maydis,Cercospora sorghi, Ustilago maydis, Puccinia spp., Macrophominaphaseolina, Penicillium oxalicum, Nigrospora oryzae, Cladosporiumherbarum, Curvularia spp., Clavibacter michiganense subsp. nebraskense,Trichoderma viride, Claviceps sorghi, Pseudomonas avenae, Corn stuntspiroplasma, Diplodia macrospora, Sclerophthora macrospora,Peronosclerospora spp., Sphacelotheca reiliana, Physopella zeae,Cephalosporium spp., Maize Dwarf Mosaic Virus A & B, Wheat Streak MosaicVirus, Maize Chlorotic Dwarf Virus, Maize Chlorotic Mottle Virus, HighPlains Virus, Maize Mosaic Virus, Maize Rayado Fino Virus, Maize StreakVirus, Maize Stripe Virus, Maize Rough Dwarf Virus; Sorghum: Exserohilumturcicum, Colletotrichum graminicola (Glomerella graminicola),Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina,Pseudomonas spp., Xanthomonas campestris p.v. holcicola, Pucciniapurpurea, Macrophomina phaseolina, Periconia circinata, Fusarium spp.,Alternaria alternata, Bipolaris sorghicola, Helminthosporium sorghicola,Curvularia lunata, Phoma insidiosa, Ramulispora spp., Phyllacharasacchari, Sporisorium spp., Sphacelotheca cruenta, Sugarcane mosaic H,Maize Dwarf Mosaic Virus A & B, Claviceps sorghi, Rhizoctonia solani,Acremonium strictum, Sclerophthona macrospora, Peronosclerospora spp.,Sclerospora graminicola, Pythium spp., etc.

Nematodes include parasitic nematodes such as root-knot, cyst, andlesion nematodes, including Heterodera and Globodera spp.; particularlyGlobodera rostochiensis and Globodera pailida (potato cyst nematodes);Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beetcyst nematode); and Heterodera avenae (cereal cyst nematode). Additionalnematodes include: Heterodera cajani; Heterodera trifolii; Heteroderaoryzae; Globodera tabacum; Meloidogyne incognita; Meloidogyne javonica;Meloidogyne hapla; Meloidogyne arenaria; Meloidogyne naasi; Meloidogyneexigua; Xiphinema index; Xiphinema italiae; Xiphinema americanum;Xiphinema diversicaudatum; Pratylenchus penetrans; Pratylenchusbrachyurus; Pratylenchus zeae; Pratylenchus coffeae; Pratylenchusthornei; Pratylenchus scribneri; Pratylenchus vulnus; Pratylenchuscurvitatus; Radopholus similis; Radopholus citrophilus; Ditylenchusdipsaci; Helicotylenchus multicintus; Rotylenchulus reniformis;Belonolaimus spp.; Paratrichodorus anemones; Trichodorus spp.;Primitivus spp.; Anguina tritici; Bider avenae; Subanguina radicicola;Tylenchorhynchus spp.; Haplolaimus seinhorsti; Tylenchulussemipenetrans; Hemicycliophora arenaria; Belonolaimus langicaudatus;Paratrichodorus xiphinema; Paratrichodorus christiei; Rhadinaphelenchuscocophilus; Paratrichodorus minor; Hoplolaimus galeatus; Hoplolaimuscolumbus; Criconemella spp.; Paratylenchus spp.; Nacoabbus aberrans;Aphelenchoides besseyi; Ditylenchus angustus; Hirchmaniella spp.;Scutellonema spp.; Hemicriconemoides kanayaensis; Tylenchorynchusclaytoni; and Cacopaurus pestis.

The methods of the embodiments can be used with other methods availablein the art for enhancing disease and pathogen resistance in plants.Similarly, the antimicrobial compositions described herein may be usedalone or in combination with other nucleotide sequences, polypeptides,or agents to protect against plant diseases and pathogens. Although anyone of a variety of second nucleotide sequences may be utilized,specific embodiments of the invention encompass those second nucleotidesequences that, when expressed in a plant, help to increase theresistance of a plant to pathogens.

Proteins, peptides, and lysozymes that naturally occur in insects(Jaynes et al., (1987) Bioassays 6:263-270), plants (Broekaert et al.(1997) Critical Reviews in Plant Sciences 16:297-323), animals (Vunnamet al., (1997) J. Peptide Res. 49:59-66), and humans (Mitra and Zang(1994) Plant Physiol. 106:977-981; Nakajima et al., (1997) Plant CellReports 16:674-679) are also a potential source of plant pathogenresistance (Ko, K. (2000) on the world wide web atScisoc.org/feature/BioTechnology/antimicrobial.html). Examples of suchplant resistance-conferring sequences include those encoding sunflowerrhoGTPase-Activating Protein (rhoGAP), lipoxygenase (LOX), AlcoholDehydrogenase (ADH), and Sclerotinia-Inducible Protein-1 (SCIP-1)described in U.S. application Ser. No. 09/714,767, herein incorporatedby reference. These nucleotide sequences enhance plant diseaseresistance through the modulation of development, developmentalpathways, and the plant pathogen defense system. It is recognized thatsuch second nucleotide sequences may be used in either the sense orantisense orientation depending on the desired outcome.

In another embodiment, the cyclotides comprise isolated polypeptides.The cyclotides of the embodiments find use in the decontamination ofplant pathogens during the processing of grain for animal or human foodconsumption; during the processing of feedstuffs, and during theprocessing of plant material for silage. In this embodiment, thecyclotides are presented to grain, plant material for silage, or acontaminated food crop, or during an appropriate stage of the processingprocedure, in amounts effective for antimicrobial activity. Thecompositions can be applied to the environment of a plant pathogen by,for example, spraying, atomizing, dusting, scattering, coating orpouring, introducing into or on the soil, introducing into irrigationwater, by seed treatment, or dusting at a time when the plant pathogenhas begun to appear or before the appearance of pests as a protectivemeasure. It is recognized that any means that bring the cyclotidepolypeptides in contact with the plant pathogen can be used in thepractice of the embodiments.

Additionally, the compositions can be used in formulations used fortheir antimicrobial activities. Methods are provided for controllingplant pathogens comprising applying a decontaminating amount of apolypeptide or composition of the embodiments to the environment of theplant pathogen. The polypeptides of the embodiments can be formulatedwith an acceptable carrier into a composition(s) that is, for example, asuspension, a solution, an emulsion, a dusting powder, a dispersiblegranule, a wettable powder, an emulsifiable concentrate, an aerosol, animpregnated granule, an adjuvant, a coatable paste, and alsoencapsulations in, for example, polymer substances.

Such compositions disclosed above may be obtained by the addition of asurface-active agent, an inert carrier, a preservative, a humectant, afeeding stimulant, an attractant, an encapsulating agent, a binder, anemulsifier, a dye, a UV protectant, a buffer, a flow agent orfertilizers, micronutrient donors or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bacteriocides, nematocides,molluscicides, acaracides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants, or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular targetmycotoxins. Suitable carriers and adjuvants can be solid or liquid andcorrespond to the substances ordinarily employed in formulationtechnology, e.g., natural or regenerated mineral substances, solvents,dispersants, wetting agents, tackifiers, binders, or fertilizers. Theactive ingredients of the embodiments are normally applied in the formof compositions and can be applied to the crop area or plant to betreated, simultaneously or in succession, with other compounds. In someembodiments, methods of applying an active ingredient of the embodimentsor an agrochemical composition of the embodiments (which contains atleast one of the proteins of the embodiments) are foliar application,seed coating, and soil application.

Suitable surface-active agents include, but are not limited to, anioniccompounds such as a carboxylate of, for example, a metal; a carboxylateof a long chain fatty acid; an N-acylsarcosinate; mono or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecylsulfate, or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates;ethoxylated alkylphenol sulfates; lignin sulfonates; petroleumsulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates orlower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;salts of sulfonated naphthalene-formaldehyde condensates; salts ofsulfonated phenol-formaldehyde condensates; more complex sulfonates suchas the amide sulfonates, e.g., the sulfonated condensation product ofoleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g.,the sodium sulfonate or dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g. polyoxyethylene sorbitar fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as 2, 4, 7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate, oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials include, but are not limited to, inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates, or botanical materials such as cork, powdered corncobs,peanut hulls, rice hulls, and walnut shells.

The compositions of the embodiments can be in a suitable form for directapplication or as a concentrate of a primary composition, which requiresdilution with a suitable quantity of water or other diluent beforeapplication. The decontaminating concentration will vary depending uponthe nature of the particular formulation, specifically, whether it is aconcentrate or to be used directly.

In a further embodiment, the compositions, as well as the polypeptidesof the embodiments, can be treated prior to formulation to prolong theactivity when applied to the environment of a plant pathogen as long asthe pretreatment is not deleterious to the activity. Such treatment canbe by chemical and/or physical means as long as the treatment does notdeleteriously affect the properties of the composition(s). Examples ofchemical reagents include, but are not limited to, halogenating agents;aldehydes such as formaldehyde and glutaraldehyde; anti-infectives, suchas zephiran chloride; alcohols, such as isopropanol and ethanol; andhistological fixatives, such as Bouin's fixative and Helly's fixative(see, for example, Humason (1967) Animal Tissue Techniques (W.H. Freemanand Co.)).

In an embodiment of the invention, the compositions of the embodimentscomprise a microbe having stably integrated the nucleotide sequence of acyclotide agent. The resulting microbes can be processed and used as amicrobial spray. Any suitable microorganism can be used for thispurpose. See, for example, Gaertner et al. (1993) in Advanced EngineeredPesticides, Kim (Ed.). In one embodiment, the nucleotide sequences ofthe embodiments are introduced into microorganisms that multiply onplants (epiphytes) to deliver the cyclotides to potential target crops.Epiphytes can be, for example, gram-positive or gram-negative bacteria.

It is further recognized that whole, i.e., unlysed, cells of thetransformed microorganism can be treated with reagents that prolong theactivity of the polypeptide produced in the microorganism when themicroorganism is applied to the environment of a target plant. Asecretion signal sequence may be used in combination with the gene ofinterest such that the resulting enzyme is secreted outside themicroorganism for presentation to the target plant.

In this manner, a gene encoding a cyclotide agent of the embodiments maybe introduced via a suitable vector into a microbial host, and saidtransformed host applied to the environment, plants, or animals.Microorganism hosts that are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one ormore crops of interest may be selected for transformation. Thesemicroorganisms are selected so as to be capable of successfullycompeting in the particular environment with the wild-typemicroorganisms, to provide for stable maintenance and expression of thegene expressing the detoxifying polypeptide, and for improved protectionof the proteins of the embodiments from environmental degradation andinactivation.

Such microorganisms include bacteria, algae, and fungi. Illustrativeprokaryotes, both Gram-negative and -positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiaceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae; and Nitrobacteraceae.Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, whichincludes yeast, such as Saccharomyces and Schizosaccharomyces; andBasidiomycetes yeast, such as Rhodotorula, Aureobasidium,Sporobolomyces, and the like.

Of particular interest are microorganisms, such as bacteria, e.g.,Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces,Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., Saccharomyces, Pichia, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, Aureobasidium, andGliocladium. Of particular interest are such phytosphere bacterialspecies as Pseudomonas syringae, Pseudomonas fluorescens, Serratiamarcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonasspheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenesentrophus, Clavibacter xyli, and Azotobacter vinlandii; and phytosphereyeast species such as Rhodotorula rubra, R. glutinis, R. marina, R.aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomycesroseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pullulans.

The cyclotides of the embodiments can be used for any applicationincluding coating surfaces to target microbes. In this manner, targetmicrobes include human pathogens or microorganisms. Surfaces that mightbe coated with the cyclotides of the embodiments include carpets andsterile medical facilities. Polymer bound polypeptides of theembodiments may be used to coat surfaces. Methods for incorporatingcompositions with antimicrobial properties into polymers are known inthe art. See U.S. Pat. No. 5,847,047 herein incorporated by reference.

The embodiments are further defined in the following Examples. It shouldbe understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of the embodiments, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the embodiments to adapt them to various usages andconditions. Thus, various modifications of the embodiments, in additionto those shown and described herein, will be apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

All publications, patents and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications, patents and patentapplications are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

EXAMPLES Example 1 Extraction and Isolation of Plant Cyclotides

Tissue from Viola spp. (8.0 g, wet weight) was harvested from plantsgrown in a growth chamber under standard conditions. The Viola spp.tissue was ground and extracted with buffer (50 mM Na₂HPO₄, 50 mMNaH₂PO₄, 50 mM Tris-HCl, 100 mM KCl, 2 mM EDTA). The crude extract wasfiltered through a cotton-plug filter to remove plant debris while fineparticulate matter was removed by centrifugation (Sorvall® InstrumentsRC5C, 15,000 rpm, 15 minutes, 4° C.). The supernatant was partitionedwith n-Butanol (BuOH). The BuOH layer was dried in a speedvac andredissolved in 2 mL distilled water. The sample (100 μL/run) wasfractionated by reverse phase high performance liquid chromatography,RP-HPLC.

Example 2 Fractionation by RP-HPLC

RP-HPLC was performed on a Hewlett-Packard HP1100 series using a Vydac®300 angstrom pore size, 10 microns particle C18 column (catalog number218TP104, Grace Vydac, W.R. Grace & Co., Columbia, Md.) and a 0-80%gradient from Solvent A (95% H₂O, 5% acetonitrile, 0.1% trifluoroaceticacid) to Solvent B (5% H₂O, 95% acetonitrile, 0.1% trifluoroacetic acid)over 40 minutes with a flow rate of 0.6 mL/min. Samples for bioassayagainst fungi were collected in 96 well plates on a Foxy™ 200 fractioncollector (Isco, Inc., Lincoln, Nebr.). The plates were lyophilized andassayed against different targets in replicates of two. Peptides havingspecific activity against fungi and nematodes were purified tohomogeneity on a capillary reverse phase C18 column (Magic 2002 HPLCSystem, Michrom BioResources, FUTECS, Daejon Korea) utilizing thefollowing gradient: 10-30% Solvent B over 10 minutes followed by 30-60%Solvent B over 60 minutes; and subsequently assayed in a dose-dependentor dose-response manner.

The HPLC profile of the crude extract of Viola spp. is shown in FIG. 1.The peak labeled 2 was specifically active against Panagrellusredivivus, Caenorhabditis elegans, and several fungi. It was purified tohomogeneity for dose-dependent assays and further biophysical andbiochemical characterization.

Example 3 Mass Spectrometry

Mass spectra were acquired on a Micromass Platform LCZ instrument(Waters Mass Spectrometry Systems, Micromass Division, Manchester, U.K.)during LCMS runs. Dried samples of the crude extract were dissolved indistilled water to give a concentration of approximately 1 mg/mL, and 10μL was injected into the solvent stream for introduction into theionization source of the mass spectrometer. Mass spectra were obtainedover the range 900-2200 m/z+ and processed using the software MassLynx™,version 3.1. The gradient was started at 0% buffer B (5% H₂O, 95%acetonitrile, 0.1% trifluoroacetic acid) progressing to 75% in 30minutes with a flow rate of 50 μL. The mass data of peak 2 indicated thepresence of two cyclotides with the deconvoluted masses of 3124.8 Da and3151.8 Da.

Example 4 Bioactivity of Cyclotide 2 Against the Nematode Panagrellusredivivus

Panagrellus was grown in basal medium and diluted in sterile water tocreate a stock solution concentration of 200 nematodes/50 μL (the basalmedia inoculum). Using a multi-channel pipette, 50 μL of the nematodestock solution was added to each well of plates containing thefractionated crude extract. Tests were performed on duplicate samples.Once the activity was established, the HPLC peaks corresponding to theactive wells were purified and dose-response assays were done.

Samples for a dose-response assay were resuspended in 40 μL and 4 wellsof 8 μL, 6 μL, 4 μL and 2 μL were prepared in replicates of two. Thevolume was adjusted with the basal media inoculum to get a final volumeof 50 μL and 200 nematodes per well. The results (see Table 1) indicatedthat cyclotide 2 was active against P. redivivus with a LD₉₀concentration (concentration at which 90% of the nematodes are killedwithin 24 hrs observation time) at 40.27 μM. The raw data for this assayis shown in Table 1. The percent mortality was calculated as ratio ofdead nematodes against the total (living and dead) number present in thetest wells. TABLE 1 Raw score for the nematocidal activities ofcyclotide 2 Concentrations (μM) 60.40 50.34 40.27 30.20 20.13 10.07Scored at 18 hrs 90 85 85 70 >60 60 (% mortality) Scored at 24 hrs ˜10095 90 80 78 74 (% mortality)

Example 5 Bioactivity of Cyclotide 2 Against Fungal Pathogens andFilamentous Fungi

The anti-fungal activity of cyclotide 2 was tested against the croppathogens Fusarium verticilloides and Colletotrichum graminaria, and thefilamentous fungus Neurospora crassa. To conduct the assay, 100 μL of ½strength potato dextrose broth (PDB) containing a spore suspension of2500 spores/mL was added to each well in a 96 well microtiter platecontaining dried HPLC fractions. The plates were mixed three times inapproximately five minute intervals before incubation at 28° C., andwere scored after 24 hours and 48 hours for inhibition of fungal growth.Inhibition of fungal growth was defined as little to no sporegermination with no detectable hyphae growth.

The dose-response fungal assays were performed by resuspending thelyophilized cyclotide protein sample in 200 μL of a solution whichconsisted of fungal spores suspended in ½ strength potato dextrose broth(PDB) at a concentration of 2500 spores/mL. The spores were either fromFusarium verticilloides, Colletotrichum graminaria or Neurospora crassa.This resuspended sample represented a starting stock solution. A 0.5×dilution series was then prepared by removing 100 μL of the startingstock solution and adding it to a well in a 96 well tray containing 100μL of spore suspension (2500 spores/mL), mixing thoroughly and thentransferring 100 μL of the newly diluted cyclotide/spore suspension to afresh well containing 100 μL of spore suspension etc., until all 12columns of the row of sample in the 96 well tray were prepared. Thefinal volume of the assay was then 100 μL. The plates were mixed threetimes in approximately five minute intervals before they were incubatedat 28° C. and then were scored after 24 and 48 hours for inhibition offungal growth. Inhibition of fungal growth was defined as little to nospore germination with no detectable hyphae growth. The results of theassays are shown in Table 2. TABLE 2 Cyclotide concentration (μM) atwhich total inhibition of fungal growth is observed. Cyclotide 2 Fungiconcentration (μM) Colletotrichum 2.5 Fusarium 10 Neurospora 1.25

Example 6 Bioactivity of Cyclotide 2 Against Scierotinia scierotiorum

To test the activity of cyclotide 2 against Sclerotinia, a Sclerotiniatest inoculum was started by inoculating ½ strength PDB liquid with asterile loop of hyphae from a Sclerotinia culture propagated on a ⅛strength potato dextrose agar (PDA) plate. This liquid inoculum was heldat room temperature (22° C.), without shaking, in the dark for 4 days,to allow sufficient growth of hyphae. The resulting suspension wasmacerated using a sterile polytron tissue grinder. The sample was thendiluted with ½ strength PDB to the point of invisibility to the nakedeye (observation under microscopy at 40× indicated the presence of 12-15hyphal fragments per 50 μL aliquot of inoculum). Then, 50 μL of theinoculum was added to each well of a 96 shallow well bioassay plate.

The cyclotide sample, following fractionation and lyophilization, wasresuspended in 100 μL ½ strength PDB, placed on ice and gently shaken.Then, 50 μL of resuspended sample was added to each corresponding wellof the bioassay plate to give a final test volume of 100 μL and 6-8hyphal fragments per well. In the inoculum control wells, 50 μL ofinoculum and 50 μL of ½ strength PDB were used, while the media controlwells contained 100 μL of ½ strength PDB. The wells were covered withBreathe-Easy membranes (Web Scientific Ltd., Cheshire, U.K.) and placedin the dark on the benchtop. The plates were evaluated 24 hours and 48hours post-inoculation and activity was demonstrated by inhibition ofhyphal growth.

For the dose-response assay, lyophilized cyclotide 2 was resuspended in40 μL of ½ strength PDB solution. Then, 20 μL of the resuspended samplewas pipetted into 4 different wells containing either 8 μL, 6 μL, 4 μLor 2 μL of suspension. The final volume in each well was adjusted to 100μL with the Sclerotinia test inoculum. The hyphal suspension was addedto each well resulting in the final concentrations listed in Table 3.The plates were covered with Breathe-Easy membranes (Web ScientificLtd., Cheshire, U.K.) and placed in the dark on the benchtop. The plateswere evaluated 24 hours and 48 hours post-inocculation and activity wasdemonstrated by inhibition of hyphal growth at 48 hours. The results ofthis assay are shown in Table 3. TABLE 3 Results of Sclerotinia assaysagainst cyclotide 2 Cyclotide 2 concentration (μM) inoculum media 6.675.0 3.33 1.67 control control Score 3 4 2 1 0 4Score index:0 = no inhibition of fungal growth1 = slight inhibition2 = moderate inhibition3 = extensive inhibition4 = total inhibition

Example 7 Bioactivity of Cyclotide 2 Against the Nematode Caenorhabditiselegans

Samples of cyclotide 2 to be used in a dose-response assay wereresuspended in 180 μl distilled H₂O to create a stock solution of 161.3μM. The dose-response assay was carried out in 96 well microtiter plateswith each dose prepared in replicates of two. The concentrations foreach dose are listed in Table 4. Each assay well contained 50 L1-stagenematodes which had been grown in S-medium (100 mM NaCl, 10 mM Kcitrate, pH 6.0, 50 mM KHPO₄, pH 6.0, 3 mM CaCl₂, 3 mM MgCl₂, 50 mMEDTA, 25 mM FeSO₄, 10 mM MnCl₂, 10 mM ZnSO₄, 1 mM CuSO₄) and had beenallowed to feed on overnight cultured E. coli strain OP50, 30 μg/mLtetracycline and 30 μg/mL chloramphenicol. The total assay volume was100 μL. The assay was scored at days 3 and 4 (Table 3). The LD₉₀recorded after day three was 6.48 μM. TABLE 4 Nematocidal activity ofcyclotide 2 against C. elegans Replicate 1 Replicate 2 ConcentrationConcentration Replicate 1 Replicate 2 (μM) (μM) Score Score A 0.81 0.8110 10 B 0.81 0.81 10 10 C 1.62 1.62 10 10 D 1.62 1.62 10 10 E 3.24 3.245 5 F 3.24 3.24 5 5 G 6.48 6.48 2 2 H 16.2 16.2 1 1Scoring Index1 - no development2 - little development5 - medium development10 - full development

Example 8 Production of Viola spp. cDNA Libraries

Total RNA from Viola spp. leaves was prepared by pulverizing the tissuewith a mortar and pestle in liquid nitrogen and lysing cells in thepresence of TRIzol™ (Invitrogen Life Technologies, Carlsbad, Calif.)according to the manufacturer's protocol. Viola leaves were harvesteddirectly into liquid nitrogen before processing. PolyA(+) RNA wasoligo(dT)-cellulose affinity column purified from total RNA using themRNA Purification Kit (Amersham Pharmacia Biotech, CA) and following thekit's protocol in preparation for cDNA library construction. Firststrand cDNA synthesis was performed using Superscript II™ (InvitrogenLife Technologies) and subsequent second strand synthesis, linkeraddition, and directional cloning into the EcoRI and XhoI sites ofpBlueScript™ SK+ (Stratagene, La Jolla, Calif.) was performed accordingto the instructions provided with the Stratagene cDNA kit (Stratagene).cDNA was purified using a cDNA column (Invitrogen Life Technologies)immediately prior to ligation into the vector.

Sequencing of cDNA library clones was performed using the ABI PRISM™ BigDye Terminator Cycle Sequencing Ready reaction kit with FS AmpliTaq™ DNApolymerase (Applied Biosystems, Foster City, Calif.) and analyzed on anABI Model 373 Automated DNA Sequencer (Applied Biosystems).

Example 9 N-Terminal Sequencing

Approximately 1.0 μg of cyclotide 2 was reduced with TCEP and alkylatedwith maleimide. It was subsequently cleaved with Endo-Glu C to yield alinear chain peptide. The mass of the peptide was monitored at eachstage. The N-terminal tag of the cleaved species was sequenced using anautomatic Edman sequencer (494 Protein Sequencer, Applied Biosystems,Foster City, Calif.) for 11 cycles. Peptide sequences corresponding tothose obtained by amino acid sequencing of the Endo-GluC treated activeswere used to compare to the corresponding cDNA clone sequence librarytranslated in all 6 reading frames using TBLASTN or TFASTA programs.Sequences containing 100% identity to the experimentally generated aminoacid sequence(s) were fully translated and their predicted molecularweight (MW) compared to the MW of purified active protein. Sequenceswith comparable MWs (within error limits) were identified as those thatencoded the peptide of interest.

The following partial sequence was obtained: N-terminal sequence (SEQ IDNO:5): SCVWIPCISAA

The above N-terminal tag could correspond to two sequences in the cDNAlibraries constructed from the Viola spp. SEQ ID NO:6 GIPCG ESCVW IPCISAAIGC SCKSK VCYRN Mass = 3124.75 SEQ ID NO:7 GIPCG ESCVW IPCIS AAIGCSCKNK VCYRN Mass = 3151.78

SEQ ID NO:6 has the same predicted mass as cyclotide 2 (3124.78 vs3124.75), it thus represents the sequence of cyclotide 2. A peptide witha mass of 3151.8 Da (cf: 3151.78) was observed to co-elute withcyclotide 2 from the crude extract, however it was a very minorcomponent (approximately 10% intensity). SEQ ID NO:6 differs from SEQ IDNO:7 only at the 24^(th) position where it has a serine instead of anasparagine. Both sequences have not previously been described in theliterature. There are, however, three other cyclotides, the circulinpeptides A and F and cycloviolacin 06, which could have the sameN-terminal tags if cleaved at the same position with Endo Glu C (see thesequences listed below).

Sequences of cyclotides with similar N-terminal tags are as follows:circulin A (SEQ ID NO:8): GIPCGESCVWIPCISAALGCSCKNKCYRN Mass = 3151.78(Gustafson et al. (1994) J. Am. Chem. Soc. 116, 9337—9338) circulin F(SEQ ID NO:9): AIPCGESCVWIPCISAAIGCSCKNKVCYR Mass = 3051.70 (Gustafsonet al. (2000) supra) cyclo. O6 (SEQ ID NO:10):GTLPCGESCVWIPCISAAVCGSKCSKVCYKN Mass = 3183.82 (Craik et al. (1999)supra)

Circulin F and cycloviolacin 06 are ruled out as potential candidatesbecause their mass clearly violates the observed mass of the twobioactive cyclotides. Circulin A has the mass of 3151.78 Da and agreeswell with the observed mass of the second peptide. However, there is nocorresponding cDNA sequence in the libraries that encodes Circulin A.

1. An isolated cyclotide-like polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 2, 4, 6 or
 7. 2. The polypeptide ofclaim 1, wherein said polypeptide is characterized by anti-fungalactivity against at least one plant fungal pathogen.
 3. The polypeptideof claim 2, wherein said plant fungal pathogen is Sclerotiniasclerotiorum.
 4. The polypeptide of claim 1, wherein said polypeptide ischaracterized by pesticidal activity against at least one species ofnematode.
 5. The polypeptide of claim 4, wherein said species ofnematode is Panagrellus redivivus.
 6. The polypeptide of claim 4,wherein said species of nematode is C. elegans.